I would like to know more about Protein Quatitation methods. I have to measure the low protein concentration that is 10ng/ml. Presently am using Nano Orange protein quantitation kit and with this we can measure the protein concentration of 100ng or more. Kindly help me regarding this are there anybody are measuring the protein concentrations in lower concentrations with anyother means.

My sample is single skeletal muscle fiber, that i can store in 8M urea buffer.

Regards,Sudhakar.

-sudhakaraare-

i'm not sure if it is more or less sensitive than nano orange but have you tried fluorescamine.

you can also do a micro-kjeldahl nitrogen determination.

-mdfenko-

Hi,

I have question regarding protein quantification. Can anybody tell me how to roughly estimate how many cells needed to produce about 500ug-1000ug protein? Will I get this amount of protein if I culture the cells in 6-well plate, then lyse the cells when it become confluent? How do we know whether the concentration range of the protein standard is suitable for the quantification of protein concentration of sample?

Thanks in advance!

Regards,YY

-sasoriza-

QUOTE (sasoriza @ Oct 16 2008, 06:18 PM)

Hi,

I have question regarding protein quantification. Can anybody tell me how to roughly estimate how many cells needed to produce about 500ug-1000ug protein? Will I get this amount of protein if I culture the cells in 6-well plate, then lyse the cells when it become confluent? How do we know whether the concentration range of the protein standard is suitable for the quantification of protein concentration of sample?

Thanks in advance!

Regards,YY

a confluent HeLa T75 flask gives me 5-10ug protein depending on how much RIPA lysis buffer I use. would that help?

for some proteins which are in low amounts a 6 well plate wouldn't be enough.

-Curtis-

QUOTE (Curtis @ Oct 18 2008, 08:53 AM)

QUOTE (sasoriza @ Oct 16 2008, 06:18 PM)

Hi,

I have question regarding protein quantification. Can anybody tell me how to roughly estimate how many cells needed to produce about 500ug-1000ug protein? Will I get this amount of protein if I culture the cells in 6-well plate, then lyse the cells when it become confluent? How do we know whether the concentration range of the protein standard is suitable for the quantification of protein concentration of sample?

Thanks in advance!

Regards,YY

a confluent HeLa T75 flask gives me 5-10ug protein depending on how much RIPA lysis buffer I use. would that help?

for some proteins which are in low amounts a 6 well plate wouldn't be enough.

May I know how much RIPA lysis buffer u used? If use more lysis buffer, it will diluted the protein concentration. If use less volume, then can obtain more concentrated protein?

-sasoriza-

QUOTE (sasoriza @ Oct 20 2008, 08:18 PM)

May I know how much RIPA lysis buffer u used? If use more lysis buffer, it will diluted the protein concentration. If use less volume, then can obtain more concentrated protein?

for a confluent T75 HeLa flask 300-500ul RIPA is a good start.

-Curtis-

Hi, I want to ask something regarding the calculation for protein quantification. When do protein assay, we will plot a standard curve for BSA, it will be a linear line (y=mx+c). So how to calculate the protein concentration in the sample? Is it by putting the absorbance value of unknown sample into the linear line equation? Is it matter if the protein concentration of the sample not fall within the range of BSA standard concentration?

Then let say if I already calculate the concentration of protein in my sample which is 100 ug, then I just need to take 40 ug protein for enzyme reaction, so I have to calculate how much volume I need to take from the lysate right?

Let say I pipette out 10ul lysate which contain 40 ug protein into a well in 96 well plate, then I add in 90 ul buffer, the protein concentration will still be 40 ug right?

Please correct me if Im wrong....thanks in advance.

Regards,YY

-sasoriza-

QUOTE (sasoriza @ Oct 23 2008, 09:20 AM)

Hi, I want to ask something regarding the calculation for protein quantification. When do protein assay, we will plot a standard curve for BSA, it will be a linear line (y=mx+c). So how to calculate the protein concentration in the sample? Is it by putting the absorbance value of unknown sample into the linear line equation? Is it matter if the protein concentration of the sample not fall within the range of BSA standard concentration?

you calculate the line and put the reading into the line calculation and get the value. you should work with concentrations within the range of your standards.

QUOTE

Then let say if I already calculate the concentration of protein in my sample which is 100 ug, then I just need to take 40 ug protein for enzyme reaction, so I have to calculate how much volume I need to take from the lysate right?

100ug is an amount, not a concentration. concentration is the amount in a given volume. you determine how much to take based on the concentration.

QUOTE

Let say I pipette out 10ul lysate which contain 40 ug protein into a well in 96 well plate, then I add in 90 ul buffer, the protein concentration will still be 40 ug right?

your starting concentration appears to be 4ug/ul. the amount added will be 40ug, the concentration will be 0.4ug/ul.

-mdfenko-

your starting concentration appears to be 4ug/ul. the amount added will be 40ug, the concentration will be 0.4ug/ul.[/quote]

Oh, thanks for your post here!So if I use M1V1 = M2V2, for example, M1 = 100 ug/ml, V1 =?, M2= 40 ug/ml, V2 = 200 ul.So my V1 will be 80 ul, then add in 40 ul of my substract to start reaction and top up to 200 ul by adding 80 ul of buffer.

Is this correct?

-sasoriza-

QUOTE (sasoriza @ Oct 23 2008, 11:35 PM)

So if I use M1V1 = M2V2, for example, M1 = 100 ug/ml, V1 =?, M2= 40 ug/ml, V2 = 200 ul.So my V1 will be 80 ul, then add in 40 ul of my substrate to start reaction and top up to 200 ul by adding 80 ul of buffer.

Is this correct?

appears to be.

although, i would put in the buffer first and start the reaction by addition of either the substrate or enzyme (i prefer to start the reaction by addition of the enzyme).